Encyclopedia of Espionage, Intelligence, and Security

DNA Recognition Instruments

█ AGNIESZKA LICHANSKA

DNA recognition instruments allow rapid identification of the origin of
DNA in an environmental or medical sample. Recognition of the source of
DNA is important in pathogen (disease-causing agent) identification in
public health surveillance, and diagnostic and military applications.

DNA recognition instruments utilize two main methods for DNA detection and
identification, nucleic acid hybridization r polymerase chain reaction
(PCR). Hybridization of nucleic acids allows differentiation of sequences
that differ by as little as one base pair by using high temperature washes
that remove partially matched DNA strands. Hybridization relies on the
fact that single stranded DNA reforms a double stranded helix with a
complementary strand. The method requires a single stranded target
(unlabeled) and probe (labeled with a radioactive or fluorescent tag to
detect signal). PCR-based detection in modern instruments is based on
specificity provided by primers required for DNA amplification and
fluorescent probes to detect the product in real time.

A technician places a gene chip into one of the photo lithography
machines shown at a production facility in California. Gene chips are
dime-sized pieces of glass infused with DNA fragments that allow
researchers to study how and why genes react to various stimuli.

AP/WIDE WORLD PHOTOS

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New technologies for DNA recognition.
The standard methods used in diagnostics are not rapid enough for the
immediate identification of pathogens in a case of a biological attack
either on military personnel or civilians. Engineers and biologists,
therefore, are designing new technologies to make DNA recognition rapid,
robust, with increased sensitivity of the assays and improved
identification of positive samples. Optical identification methods are
primarily used in PCR-based instruments; however, new magnetic and
electrochemical methods were developed for hybridization-based assays.

Hybridization-based technologies.
Chip-based hybridization assays, where the target DNA is spotted onto a
glass or plastic slide and a single stranded DNA probe is used to detect
it, were developed recently by a number of companies. Technology allows
placement of thousands of DNA molecules on the slide, but detection of the
specific reaction is often lacking sensitivity. As a result, a number of
research teams and commercial companies are researching better ways to
identify a positive signal.

One breakthrough came with the implementation of electrical conductivity
as a detection method. This method relies on the use of electrodes with
gaps of 30–50nm in size, containing single stranded DNA molecules
(oligonucleotides) immobilized on their surface (capture probes) and gold
oligonucleotide nanoparticles allowing detection of electrical currents
resulting from hybridization. Both oligonucleotides bind to the target
sequence when the electrode is immersed in a solution containing target
molecules. A modification of this method is the use of signal
amplification by using a photographic solution as developed by a
Northwestern University team. A salt wash before the addition of
photographic developer removes mismatches and the silver coated gold
particles can be easily visualized. The chip is then scanned using a
flatbed scanner, removing the need for expensive equipment. This method is
highly sensitive and very fast. It is able to detect concentrations of DNA
(100 times more sensitive than conventional detection methods), in one to
three minutes.

A modification of this method was developed in 2002 and incorporates
nanoparticle probes that in addition to gold particles, have Raman
dye-label (for example Cy3, Cy5, or Texas Red). Detection of these probes
can be either by Raman spectroscopy or by using a flatbed scanner to
detect silver enhancement. By using multiple labels one is able to design
chips detecting multiple target sequences (multiple pathogens).

Hybridization-based instruments.
The great advantage of hybridization-based instruments is the fact that
they do not require any DNA amplification, are highly sensitive and give
rapid results.

Scientists in industry are currently producing instruments that are based
on measuring electrical conductivity. One is known as the eSensor. The
system consists of bioelectronic chips, reader, and special software. The
chips contain capture probes and signaling probes. After an interaction
with a target sequence, signaling probes induce electric current, which is
detected and interpreted by the sensor's software. This instrument
can perform a number of assays simultaneously. A second instrument is
directly based on the technology from the Northwestern University group,
using a method of conductivity detection that was modified to amplify the
signal from gold particles by using a photographic developer solution to
coat the gold particles. Although this instrument currently requires a
large space, work is underway to design a handheld device.

One company has licensed a Strand Displacement Amplification (SDA) method,
and has devised an electrical method of binding DNA to silicon chips and
performing hybridization. SDA oligonucleotides (probes) are localized to
spots on the chip by charge and immobilized on the surface by chemical
reaction. The sample is then added to the chip and by applying an electric
current, the binding of test to the probes is highly accelerated (one to
three minutes). By reversing the charge, unbound molecules are removed and
only perfect matches remain. The entire process takes about 15 minutes.
Chips for identifying pathogens such as the bacteria responsible for
anthrax are under development.

PCR-based instruments.
The newest technologies in polymerase chain reaction (PCR)-based
instruments involve instrument miniaturization and methods for handling
and detecting multiple pathogens in multiple samples. The ability to
prepare clean PCR templates in a field is often difficult or limited.
However, the presence of various chemicals can inhibit the amplification,
giving false negative results and, in the case of an attempt to identify a
biological threat, possibly endanger people's lives. As a result, a
number of companies have started to offer sample preparation units with
their PCR instruments.

The advanced nucleic acid analyzer (ANAA), developed in 1997, was the
first DNA recognition instrument designed for work in the field. It was
portable, but still large and was superseded by a hand-held ANAA (HANAA).

The major differences between the various instruments are in the
proprietary heating and cooling systems, detection optics, and sample
preparation and handling, as well as size. Speed of most of these
instruments is similar with the typical sample analysis taking 7–20
minutes.

A different technology, but still PCR-based, uses a high-performance
liquid chromatography to separate the PCR products and identify mutations.
The advantage of the system is that it can detect mutations in any genes
that could have been altered for designing biological weapons, thus,
potentially complementing any other detection methods.

Application of DNA recognition instruments.
DNA recognition instruments are likely to be used in general monitoring
of the environment, investigation of suspicious objects, and in
diagnostics. In all of these applications, detection must be rapid and
accurate in order to introduce prevention measures or rapid treatment.
Ease of use and result interpretation are important, as in majority of
cases, users will be people with minimal laboratory training.

As of 2003, the majority of these advanced DNA recognition instruments
were or are undergoing final testing in the field. They are able to cope
with samples of water, food, and various clinical samples to detect an
environmental contamination or identify a pathogen causing unusual
symptoms in humans or domestic animals.